Characterization of leukotoxin from a clinical strain of Actinobacillus actinomycetemcomitans

Diaz, Roger; Ghofaily, Lourdes Al; Patel, Jignesh; Balashova, Nataliya V.; Freitas, Anna Cristina de; Labib, Irene; Kachlany, Scott C.

doi:10.1016/j.micpath.2005.10.005

Cited by 47 publications

(59 citation statements)

References 51 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…After approximately 16 h of growth, bacterial cells were separated from the supernatant by centrifugation. Equal amounts of cell-associated and secreted protein (supernatant) were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis using antileukotoxin antibody (9). Figure 2 shows that without added FeCl 3 , LtxA is predominantly secreted, while a small amount remains associated with cells.…”

mentioning

confidence: 99%

“…To confirm that ltxA synthesis is not affected by iron, we determined the levels of ltxA mRNA produced under normal and excess-iron growth conditions by quantitative real-time PCR (qRT-PCR) using a quantitative RT-PCR ReadyMix kit according to the manufacturer's protocol (Sigma, St. Louis, MO) ( Table 1). Highly leukotoxic strains JP2 (36,41) and NJ4500, a fresh clinical isolate (9,21), were grown in AAGM broth with and without 300 M FeCl 3 . Relative to the expression of housekeeping genes glyA (29) and gapdh (34) ( Table 1), we found that the ratio of ltxA mRNA levels under excess-iron conditions to those under normal growth conditions was approximately 1 ( Table 2).…”

mentioning

confidence: 99%

See 1 more Smart Citation

Regulation of Aggregatibacter ( Actinobacillus ) actinomycetemcomitans Leukotoxin Secretion by Iron

et al. 2006

Self Cite

View full text Add to dashboard Cite

The gram-negative oral and systemic pathogen Aggregatibacter (Actinobacillus) actinomycetemcomitans produces a leukotoxin (LtxA) that is a member of the RTX (repeats in toxin) family of secreted bacterial toxins. We have recently shown that LtxA has the ability to lyse erythrocytes, which results in a beta-hemolytic phenotype on Columbia blood agar. To determine if LtxA is regulated by iron, we examined beta-hemolysis under iron-rich and iron-limiting conditions. Beta-hemolysis was suppressed in the presence of FeCl 3 . In contrast, strong beta-hemolysis occurred in the presence of the iron chelator deferoxamine. We found that secretion of LtxA was completely inhibited by free iron, but expression of ltxA was not regulated by iron. Free chromium, cobalt, and magnesium did not affect LtxA secretion. Other LtxA-associated genes were not regulated by iron. Thus, iron appears to play an important role in the regulation of LtxA secretion in A. actinomycetemcomitans in a manner independent of gene regulation.

show abstract

mentioning

confidence: 99%

mentioning

confidence: 99%

Regulation of Aggregatibacter ( Actinobacillus ) actinomycetemcomitans Leukotoxin Secretion by Iron

et al. 2006

Self Cite

View full text Add to dashboard Cite

show abstract

“…While LtxA can be associated with the outer membrane (Ohta et al, 1991;Berthold et al, 1992;Johansson et al, 2000;Diaz et al, 2006), released within lipid vesicles (Kato et al, 2002;Demuth et al, 2003), and secreted from cells as soluble protein (Brogan et al, 1994;Kachlany et al, 2000), we believe that TdeA plays a role in all of these processes. Because RTX toxins are transported through the bacterial cell envelope without a periplasmic intermediate (Gray et al, 1986;Koronakis et al, 2004), LtxA would have to be exported through TdeA before localizing to the outer membrane or within vesicles.…”

Section: Discussionmentioning

confidence: 78%

“…Proteins were transferred to nitrocellulose membranes as described previously (Sambrook et al, 1989). Immunodetection was carried out using rabbit polyclonal anti-LtxA antibody (Diaz et al, 2006) and alkaline phosphatase-conjugated goat anti-rabbit secondary antibody (Bio-Rad Laboratories, Hercules, CA). Bound antibody was detected using BCIP (5-bromo-4-chloro-3-indolylphosphate) and nitroblue tetrazolium (Bio-Rad Laboratories, Hercules, CA).…”

Section: Sds-page and Western Blot Analysismentioning

confidence: 99%

TdeA, a TolC-like protein required for toxin and drug export in Aggregatibacter (Actinobacillus) actinomycetemcomitans

Crosby

Kachlany

2007

Gene

Self Cite

View full text Add to dashboard Cite

Aggregatibacter actinomycetemcomitansW is an oral bacterium that causes localized aggressive periodontitis (LAP) and extra-oral infections such as sub-acute infective endocarditis. As part of its array of virulence factors, A. actinomycetemcomitans produces leukotoxin (LtxA), a member of the RTX family of toxins. LtxA kills human leukocytes and we have recently shown that the toxin is required for β -hemolysis by A. actinomycetemcomitans on solid medium. In other RTX toxinproducing bacteria, an outer membrane channel-forming protein, TolC, is required for toxin secretion and drug export. We have identified an ORF in A. actinomycetemcomitans that encodes a putative protein having predicted structural properties similar to TolC. Inactivation of this ORF resulted in a mutant that was no longer β -hemolytic and did not secrete LtxA. This mutant was significantly more sensitive to antimicrobial agents compared to the wild type strain and was unable to export the antimicrobial agent berberine. Thus, this ORF was named tdeA for "toxin and drug export". Examination of the DNA sequence surrounding tdeA revealed two upstream ORFs that encode proteins similar to the drug efflux proteins, MacA and MacB. Inactivation of macB in A. actinomycetemcomitans did not alter the drug sensitivity profile or the hemolytic activity of the mutant. The genes macA, macB and tdeA are organized as an operon and are constitutively expressed as a single transcript. These results show that A. actinomycetemcomitans indeed requires a TolC-like protein for LtxA secretion and that this protein, TdeA, also functions as part of a drug efflux system.

show abstract

“…Fresh clinical isolates show a small, wrinkled (rough) colony form Henderson et al, 2003;Kachlany et al, 2001a). The bacteria are known to produce several virulence factors, including a leukotoxin and a cytolethal distending toxin, and they adhere to solid surfaces and cells (Taichman and Wilton, 1981;Kolodrubetz et al, 1989;Lally et al, 1989;Eastcott et al, 1990;Mangan et al, 1991;Fives-Taylor et al, 1999;van Winkelhoff and Slots, 1999;Kachlany et al, 2001a;Narayanan et al, 2002;Henderson et al, 2003;Rose et al, 2003;Paturel et al, 2004;Rhodes et al, 2005;Diaz et al, 2006;Fine et al, 2005Fine et al, , 2006Smith and Bayles, 2006). Clinical isolates autoaggregate and secrete long, bundled pili (Flp fibrils) (Holt et al, 1980;Scannapieco et al, 1987;Rosan et al, 1988;Inoue et al, 1998;Kachlany et al, 2001b;Henderson et al, 2003;Fine et al, 2006).…”

Section: Introductionmentioning

confidence: 99%

tfoX (sxy)-dependent transformation of Aggregatibacter (Actinobacillus) actinomycetemcomitans

Bhattacharjee

Fine

Figurski

2007

Gene

View full text Add to dashboard Cite

AbstracttfoX (sxy) is a regulatory gene needed to turn on competence genes. Aggregatibacter (Actinobacillus) actinomycetemcomitans has a tfoX gene that is important for transformation. We cloned this gene on an IncQ plasmid downstream of the inducible tac promoter. When this plasmid was resident in cells of A. actinomycetemcomitans and tfoX was induced, the cells became competent for transformation. Several strains of A. actinomycetemcomitans, including different serotypes, as well as rough (adherent) and isogenic smooth (nonadherent) forms were tested. Only our two serotype f strains failed to be transformed. With the other strains, we could easily get transformants with extrachromosomal plasmid DNA when closed circular, replicative plasmid carrying an uptake signal sequence (USS) was used. When a replicative plasmid carrying a USS and cloned DNA from the chromosome of A. actinomycetemcomitans was linearized by digestion with a restriction endonuclease or when genomic DNA was used directly, the outcome was allelic exchange. To facilitate allelic exchange, we constructed a suicide plasmid (pMB78) that does not replicate in A. actinomycetemcomitans and carries a region with two inverted copies of a USS. This vector gave allelic exchange in the presence of cloned and induced tfoX easily and without digestion. Using transposon insertions in cloned katA DNA, we found that as little as 78 bp of homology at one of the ends was sufficient for that end to participate in allelic exchange. The cloning and induction of tfoX makes it possible to transform nearly any strain of A. actinomycetemcomitans, and allelic exchange has proven to be important for site-directed mutagenesis.

show abstract

Characterization of leukotoxin from a clinical strain of Actinobacillus actinomycetemcomitans

Cited by 47 publications

References 51 publications

Regulation of Aggregatibacter ( Actinobacillus ) actinomycetemcomitans Leukotoxin Secretion by Iron

Regulation of Aggregatibacter ( Actinobacillus ) actinomycetemcomitans Leukotoxin Secretion by Iron

TdeA, a TolC-like protein required for toxin and drug export in Aggregatibacter (Actinobacillus) actinomycetemcomitans

tfoX (sxy)-dependent transformation of Aggregatibacter (Actinobacillus) actinomycetemcomitans

Contact Info

Product

Resources

About